The cytochrome P-450 containing monoxygenase enzyme system is known to play key roles in the metabolism of drugs and the activation of carcinogens. This project is a continuation of our current studies on the properties of this system as well as on the mechanisms and sites of carcinogen activation. The incorporation of purified cytochrome P-450 and reductase into microsomal membrane will be studied to elucidate the mechanism of the incorporation. The effects of this incorporation on the monoxygenase activities will be analyzed to gain insight into the rate-limiting step of drug oxidations and the organization of the monoxygenase enzymes in the membrane. The enzyme topology and properties of the microsomes will be studied with cross-linking, photoinduced dichroism, and immobilization studies. Plans are made to develop fully a direct fluorescence assay for benzo(a)pyrene (BP) metabolism; to investigate the meaning of the Km of the BP hydroxylase system; to study the effects of temperature and membrane structure on monoxygenase activities; and to elucidate the mechanisms of inhibition of 7,8-benzoflavone, BHA, and other inhibitors. The biochemical and physiological effects of BHA will be studied. The binding of the metabolically activated (3H)BP to nuclear DNA, RNA, histones, and nonhistone proteins will be studied using isolated rat liver and lung nuclei. The effect of microsomes on the binding will be studied systematically to assess the role of microsomes in carcinogen activation. Similar approaches will also be used to study the sites of activation of aflatoxin B, 2-acetylaminofluorene, and CCl4. The metabolism of BP by the nuclear envelope monoxygenase system will be studied using high pressure liquid chromatography. The target molecules of the carcinogen binding will be characterized.